title: Cattle mammary bioreactor generated by a novel procedure of transgenic cloning for large-scale production of functional human lactoferrin.
abstract: Large-scale production of biopharmaceuticals by current bioreactor techniques is limited by low transgenic efficiency and low expression of foreign proteins. In general, a bacterial artificial chromosome (BAC) harboring most regulatory elements is capable of overcoming the limitations, but transferring BAC into donor cells is difficult. We describe here the use of cattle mammary bioreactor to produce functional recombinant human lactoferrin (rhLF) by a novel procedure of transgenic cloning, which employs microinjection to generate transgenic somatic cells as donor cells. Bovine fibroblast cells were co-microinjected for the first time with a 150-kb BAC carrying the human lactoferrin gene and a marker gene. The resulting transfection efficiency of up to 15.79 x 10(-2) percent was notably higher than that of electroporation and lipofection. Following somatic cell nuclear transfer, we obtained two transgenic cows that secreted rhLF at high levels, 2.5 g/l and 3.4 g/l, respectively. The rhLF had a similar pattern of glycosylation and proteolytic susceptibility as the natural human counterpart. Biochemical analysis revealed that the iron-binding and releasing properties of rhLF were identical to that of native hLF. Importantly, an antibacterial experiment further demonstrated that rhLF was functional. Our results indicate that co-microinjection with a BAC and a marker gene into donor cells for somatic cell cloning indeed improves transgenic efficiency. Moreover, the cattle mammary bioreactors generated with this novel procedure produce functional rhLF on an industrial scale.
authors.affiliation: State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing, People's Republic of China.
authors.forename: Penghua
authors.initials: P
authors.lastname: Yang
authors.forename: Jianwu
authors.initials: J
authors.lastname: Wang
authors.forename: Guochun
authors.initials: G
authors.lastname: Gong
authors.forename: Xiuzhu
authors.initials: X
authors.lastname: Sun
authors.forename: Ran
authors.initials: R
authors.lastname: Zhang
authors.forename: Zhuo
authors.initials: Z
authors.lastname: Du
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.forename: Rong
authors.initials: R
authors.lastname: Li
authors.forename: Fangrong
authors.initials: F
authors.lastname: Ding
authors.forename: Bo
authors.initials: B
authors.lastname: Tang
authors.forename: Yunping
authors.initials: Y
authors.lastname: Dai
authors.forename: Ning
authors.initials: N
authors.lastname: Li
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D019149:Bioreactors; D002417:Cattle; D017690:Cell Transplantation; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007781:Lactoferrin / Q000096:biosynthesis / Q000235:genetics*; D008321:Mammary Glands, Animal / Q000378:metabolism*; D053652:Nuclear Transfer Techniques; D011994:Recombinant Proteins; D014162:Transfection / Q000379:methods
chemical_list: D011994:Recombinant Proteins; D007781:Lactoferrin